KR100412117B1 - Methods for recovering isoflavones from soybean molasses - Google Patents

Abstract

Methods for recovering isoflavones and derivatives thereof from soy molasses are disclosed. In a first embodiment, a method is disclosed in which isoflavones are recovered without any significant conversion of isoflavone conjugates to other forms. In a second embodiment, a method is disclosed whereby isoflavone conjugates are converted to glucosides while in the soy material prior to their recovery. In a third embodiment, a method is disclosed in which isoflavones are converted to their aglucone form while in the soy material and prior to their recovery. Also disclosed are various isoflavone enriched products obtained from soy molasses.

Description

Methods for recovering isoflavones from soybean molasses

The present invention relates to a method for recovering isoflavones from soybean molasses. The present invention also relates to a product containing isoflavones.

Isoflavones are produced in a variety of soybean plants, including plant protein materials such as soybeans. These compounds include daidzin, 6 "-OAc didezin, 6" -OMal didezin, daidzein, genistin, 6 "-OAc genistin, 6" -OMal genistin , Genistein, glycitin, 6 " -OAc-glycitin, 6 " -OMal glycitin, glycitein, biochanin A, formononentin and coumestrol. Generally, these compounds cause the bitter taste of soybeans to be felt.

Isoflavones in soy material include isoflavone glucoside (glucone), isoflavone complex and aglucone isoflavone. Isoflavone glucoside has a glucose molecule attached to the isoflavone moiety. The isoflavone complex has an additional moiety attached to the glucose molecule of the isoflavone glucoside, for example, 6 " -OAc genistein contains an acetate group attached at position 6 of the glucose molecule of genistin. Isoflavones consist entirely of isoflavones.

Soybeans are a family of isoflavone compounds with the corresponding glucosides, complexes and aglucone members, namely the genistein group; Daid Jane County; And a glycitein group. The genistein group includes glucoside genistein; Complex 6 "-OMalgenitin (6" -malonate ester of genistin) and 6 "-OAc genistein (6" -acetate ester of genistin) and acluconenzynthin. The daidzein group is glucoside dideazine; Complex 6 " -OMal dideazine and 6 " -OAc dideazine; And agluconidase. ≪ / RTI > The glycitein group is glucoside glycite; Complex 6 " -OMAL glycitin, and agglutinin glycitein.

The most critical problem in the manufacture of commercial products such as vegetable protein concentrates has been the removal of these materials. For example, in a conventional method of making a soy protein concentrate, soy flakes are extracted with aqueous acid or aqueous alcohol to remove water soluble materials from soy flakes, where large amounts of isoflavones are dissolved in the extract have. The extract of water soluble material containing isoflavones is soybean molasses. The soy molasses is a by-product of the production of soy protein concentrates, which are usually discarded. Thus, it is clear that if molasses can be separated from soy molasses, soy molasses is inexpensive and is a preferred source of isoflavones.

Recently, the medical value of isoflavones contained in vegetable proteins such as soybeans has been recognized. Especially aglucone isoflavone attracts attention. Genistein and daidzein can significantly reduce cardiovascular risk factors (see " Plant and Mammalian Estrogen Effects on Plasma Lipids of Female Monkeys ", Circulation , vol. 90, p. 1259 (October 1994)]. Also. It is believed that genistein and daidzein reduce the symptoms caused by the reduction and alteration of the amount of endogenous estrogen in women, such as the symptoms of menopausal or premenstrual syndrome. Also, recently, it has been shown that aglucone isoflavones can inhibit the growth of human cancer cells, such as breast cancer cells and prostate cancer cells (Peterson and Barnes, Biochemical and Biophysical Research, Communications, Vol. 179, No. 1, pp. Quot; Genistein Inhibition of the Growth of Human Breast Cancer Cells, Independence from Estrogen Receptors and the Multi-Drug Resistance Gene " of Peterson and Barnes, The Prostate, Vol. 661-667 (August 30, 1991) 22, pp. 335-345 (1993), " Genistein and Biochanin A Inhibits the Growth of Human Prostate Cancer Cells but not Epidermal Growth Factor Receptor Tyrosine Autophosphorylation " Barnes et al. Mutagens and Carcinogens in the Diet. pp. 239-253 (1990) "Soybeans Inhibit Mammary Tumors in Models of Breast Cancer").

The structural formula of the aglycon isoflavone is shown below:

[Chemical Formula 1]

In this formula,

R ₁ , R ₂ , R ₃ and R ₄ can be selected from the group consisting of H, OH and OCH ₃ . Wherein R ₁ is OH, R ₂ is H, R ₃ is OH, and R ₄ is OH, the daidzein is a compound of formula 1 wherein R ₁ is OH, R ₂ is H, R ₃ and is H, the compounds of the formula (1) of R ₄ is OH, and glycitein is R ₁ is OH, R ₂ is OCH _3, and R ₃ is H, R ₄ is a compound of the formula (1) OH .

The present invention therefore relates to the recovery of isoflavones and soy flour from an isoflavone-rich material. The present invention further provides a process for the production of isoflavone glucoside and aglucone isoflavone, conversion of soy isanomal isoflavone to isoflavone glucoside and aglucone isoflavone, production of isoflavone glucoside rich material from soy molasses, And recovery of flavon-rich materials.

A common method for converting vegetable protein isoflavone complexes to aglucone isoflavones is known and described in copending U.S. application Serial No. 08 / 477,102, filed June 7, 1995, assigned to the assignee hereof, ≪ / RTI >

Other methods known in the art for converting isoflavone glucoside into aglucone isoflavone are described, for example, in Japanese Patent Application No. 258,669 (Obata et al.). These methods are not concerned with the recovery of isoflavone-rich material from soybean molasses. In addition, the methods do not provide a method of converting the isoflavone complex into isoflavone glucoside or aglucone isoflavone. In addition, these methods can only achieve a moderate conversion rate in converting isoflavone glucoside to aglucone isoflavone, but require significant time to achieve this intermediate conversion rate.

A first object of the present invention is to provide a method of producing an isoflavone rich material from soybean molasses and an isoflavone rich material.

A second object of the present invention is to provide a method of producing an isoflavone glucoside-rich material and an isoflavone glucoside-rich material from soybean molasses.

A third object of the present invention is to provide a material rich in aglucone isoflavone and a method of producing aglucone isoflavone rich material from soybean molasses.

The present invention relates to a method for recovering an isoflavone rich material from a soy molasses material containing an isoflavone rich material and an isoflavone. The method comprising: providing soy molasses containing isoflavones; And separating the cake from the soy molasses material at a pH and temperature sufficient to ensure that most isoflavones are contained within the cake. Preferably, the pH at the time of separation is about 3.0 to about 6.5, and the temperature is about 0 to about 35 ° C. The cake is an isoflavone-rich material.

In one embodiment, the glucoside-rich isoflavone material is formed from a cake of isoflavone-rich material. The aqueous slurry is formed of an isoflavone-rich material. The slurry is treated for a time sufficient to convert the isoflavone complex in the isoflavone rich material to isoflavone glucoside at a temperature of from about 2 DEG C to about 120 DEG C and a pH of from about 6 to about 13.5. The cake made of isoflavone glucoside-rich material can then be separated from the slurry.

In another embodiment, the aglucone isoflavone rich material is formed from a cake comprised of an isoflavone rich material. The aqueous slurry is formed of an isoflavone-rich material. The slurry is treated for a time sufficient to convert the isoflavone complex in the isoflavone rich material to isoflavone glucoside at a temperature of from about 2 DEG C to about 120 DEG C and a pH of from about 6 to about 13.5. The enzyme capable of cleaving the 1,4-glucosidic bond is then contacted with the isoflavone glucoside of the slurry, wherein the condition is selected from the group consisting of isoflavone glucoside It is sufficient time for the seed to convert to the aglycon isoflavone. A cake made of agglomerated isoflavone-rich material can be separated from the slurry.

In another aspect, the present invention relates to a process for producing an isoflavone glucoside-rich material and a process for producing the isoflavone glucoside-rich material from soybean molasses. The soy molasses is treated for a time sufficient to convert the isoflavone complex contained in soybean molasses to isoflavone glucoside at a temperature of from about 2 DEG C to about 120 DEG C and a pH of from about 6 to about 13.5. A cake consisting of an isoflavone glucoside rich material is separated from the molasses at a temperature and pH sufficient to ensure that most of the isoflavone glucoside is contained in the cake.

In another aspect, the present invention relates to a method for recovering the above-mentioned aglucone isoflavone-rich material and a material rich in said aglucone isoflavone from soybean molasses. The soy molasses is treated for a time sufficient to convert the isoflavone complex contained in soybean molasses to isoflavone glucoside at a temperature of from about 2 DEG C to about 120 DEG C and a pH of from about 6 to about 13.5. An enzyme capable of cleaving the 1,4-glucosidic bond and an isoflavone glucoside in the soybean molasses are then contacted with the isoflavone glucoside at a temperature of about 5 DEG C to 75 DEG C and a pH of about 3 to about 9, Is contacted for a time sufficient to convert it to cones isoflavone. A cake of agglomerated isoflavone rich material is separated from the molasses material at a sufficient pH and temperature to ensure that most of the agglomerated isoflavones are contained within the cake.

In carrying out the method of the present invention, the starting material is soybean molasses. Soybean molasses is a by-product of many commercial processes associated with soy or soy derivatives, such as the production of protein extracts, protein whey and protein concentrates. Therefore, it is possible to secure a large amount of soybean molasses at a relatively low price.

Generally, soy molasses is considered a soy soluble material separated from soy insoluble material by washing with alcohol or acid. Typically, soybean molasses is an aqueous mixture consisting of at least 50% solids comprising about 6% protein (based on the total weight of soybean molasses), about 3% ash, about 5% fat and about 36% carbohydrates. Soy molasses is also known in the art as a soy soluble material. As used herein, the term " soybean molasses material " refers to a composition comprising soybean molasses and / or derivatives of soy molasses, for example, soy isoflavone glucoside rich soy molasses material and agglomerated isoflavone rich soy molasses material. Accordingly, these terms are interchangeable terms.

In a preferred embodiment of the present invention, the soy molasses starting material is prepared from defatted soy flakes in which the oil has been removed by solvent extraction in a conventional manner. The defatted soy flakes are extracted with water adjusted to an acidic pH, preferably from about pH 4 to about pH 5, by the addition of one or more suitable acids such as acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid or any other suitable reagent. The ratio of acidic extract to soy flake is preferably in a weight ratio of about 16: 1 to about 20: 1. To improve the effectiveness of the extraction, the temperature of the extract may be raised above room temperature, preferably from about 32 ° C to about 55 ° C. After extraction, the soy molasses extract is removed from the soy-insoluble material.

In another embodiment, the defatted soy flakes are treated with aqueous alcohol to produce a soybean molasses starting material. The flakes are extracted with about 80% aqueous ethanol at a weight ratio of extract to soy flakes of about 16: 1 to about 20: 1. To improve the effect of extraction, the temperature of the alcohol extract is raised to room temperature or higher, preferably about 32 ° C to about 55 ° C. The soy molasses extract is then removed from the soybean insoluble material to provide soy molasses as a starting material.

Isoflavone-rich materials can be recovered from the soybean molasses starting material. The soy molasses material may be diluted with water to have a solids content of about 6 to about 13%, most preferably 13%. However, dilution of the soy molasses material is not a necessary condition in the method of the present invention, but may be diluted to facilitate processing of the relatively high viscosity soy molasses material.

During the separation process, the soy molasses material, preferably the diluted soy molasses material, is treated at the pH and temperature at which most of the isoflavones will separate from the soybean molasses. In a preferred embodiment, the soy molasses material is treated at a pH of from about 3.0 to about 6.5 and a temperature of from about 0 캜 to about 35 캜 to maximize the insolubility of isoflavones in soybean molasses. However, isoflavones can be isolated at pH values outside the preferred range and at temperatures above 35 DEG C, but such conditions are less desirable because smaller amounts of isoflavones are isolated from the soybean molasses during the separation process. The pH of the soy molasses may be adjusted using a suitable acidic or basic reagent as appropriate. It is most preferable to adjust the pH of soybean molasses to about 4.5. It is also preferred that the soy molasses is cooled or cooled to a temperature of from about 0 째 C to about 10 째 C, most preferably from about 4 째 C to about 7 째 C.

The soy molasses material then undergoes a separation process to separate the cake of isoflavone-rich material from the soy molasses material. The separation is carried out while maintaining the soy molasses material under the previously described pH and temperature conditions.

In one embodiment, a cake of isoflavone-rich material is separated by centrifuging the soybean molasses and decanting the supernatant from the cake. Centrifugation is preferably carried out at about 0 ° C to about 10 ° C at about 3,000 to about 10,000 rpm for about 30 minutes.

In another embodiment, the isoflavone rich cake may be separated from the molasses material by filtration. The filtration is preferably carried out at pH and temperature conditions already mentioned, preferably at a pH of about 4.5 and at a temperature of from about 0 [deg.] C to about 10 [deg.] C.

In another aspect of the present invention, a cake made of a material rich in isoflavone glucoside can be recovered from soybean molasses. The soybean molasses starting material is obtained as described above. Although not required, it is preferred that the soy molasses material be diluted to a solids content of about 6 to about 13%, most preferably about 13% to facilitate processing of the material.

Subsequently, a conversion operation is performed on soybean molasses to convert the isoflavone complex in soybean molasses to isoflavone glucoside. A significant portion of the isoflavones in the soybean molasses is the isoflavone complex, and thus the conversion substantially increases the amount of isoflavone glucoside in the soy molasses material. The conversion was found to be dependent on the pH and temperature of the soy molasses.

The pH range for the conversion of isoflavone complex to isoflavone glucoside is from about 6 to about 13.5. The pH of the molasses may be adjusted to a desired pH by using an appropriate acid or an acidic reagent in order to decrease the pH. The conversion of the isoflavone complex to isoflavone glucoside has been found to act as a catalyst, so it is most preferred to use a high pH for rapid conversion. The most preferred pH for the conversion of isoflavone complex to isoflavone glucoside is from about 9 to about 11.

The temperature range for converting the isoflavone complex in soybean molasses to isoflavone glucoside is from about 2 to about 120 < 0 > C. Within this temperature range, the conversion can easily occur depending on the pH of the soy molasses material. The present inventors have found that the conversion proceeds easily at lower temperatures when the pH is relatively high. For example, at a pH of about 11, the conversion occurred quickly and efficiently at a temperature of about 5 ° C to about 50 ° C. At a pH of about 9, the conversion occurred efficiently in the temperature range of about 45 ° C to about 75 ° C. If the pH of the soy molasses is relatively low, the conversion occurs at a higher temperature. For example, at a pH of about 6, the conversion occurs in a temperature range from about 80 ° C to about 120 ° C. In a preferred embodiment, the conversion occurs at about 35 캜 and at a pH of about 11. In another preferred embodiment, the conversion occurs at a temperature of about 73 캜 and a pH of about 9.

The time required for the conversion of the isoflavone complex to isoflavone glucoside is largely dependent on the temperature and pH range of the soybean molasses. The conversion time is typically about 15 minutes to several hours or more. The conversion occurs more rapidly at higher temperatures and higher pH. At pH about 9 and 73 캜, the conversion is substantially complete within about 4 hours to about 6 hours. In a most preferred embodiment, the isoflavone complex is converted to isoflavone glucoside at a pH of about 11 and at a temperature of about 35 DEG C for about 30 minutes to about 1 hour, preferably about 45 minutes.

The conversion of the isoflavone complex to isoflavone glucoside is highly efficient, at least most preferably, preferably, almost all of the isoflavone complex present is converted to isoflavone glucoside. The term " most " as used herein means a conversion of at least about 50%, and " almost all " means a conversion of at least about 80%, preferably at least about 90%.

After conversion of the isoflavone complex to isoflavone glucoside, a cake of isoflavone glucoside rich material can be separated from the molasses material. The soy molasses material is treated at the pH and temperature at which most of the isoflavone glucoside is separated from the soy molasses material during the separation process. In a preferred embodiment, the soy molasses material has a pH in the range of about 3 to about 6.5, most preferably about 4.5, and a temperature of about 0 캜 to about 35 캜, preferably about 0 캜 to about 10 캜, Lt; RTI ID = 0.0 > 4 C < / RTI > The pH of the soy molasses material may be adjusted using a suitable acidic or basic reagent as appropriate.

The separation may be carried out by conventional methods for separating solids from liquids. The isoflavone glucoside rich cake is preferably separated by centrifugation or filtration as described above in connection with separating the isoflavone-rich cake from the soy molasses material.

In another aspect of the present invention, a cake of agglomerated isoflavone rich material can be recovered from the molasses material. The soybean molasses starting material is obtained as described above and is preferably diluted with water to provide a solids content of about 6% to about 13%. The soy molasses starting material is treated as previously described to convert the isoflavone complex to isoflavone glucoside.

The enzymatic conversion procedure is then carried out by contacting the appropriate enzyme with isoflavone glucoside in a soy molasses material at an appropriate pH and temperature in an isoflavone glucoside-rich soy molasses to convert the isoflavone glucoside to an aglucone isoflavone . The two stage conversion process efficiently converts almost all of the isoflavone complex and isoflavone glucoside in the soy molasses material to aglucone isoflavone and significantly increases the amount of aglucone isoflavone in the soy molasses material.

The conversion of isoflavone glucoside to aglucone isoflavone depends on a variety of factors including the type of enzyme present in the soy molasses material, the distribution of the enzyme concentration, the activity of the enzyme, and the pH and temperature of the soy molasses material at the time of conversion Respectively. The enzyme required to carry out this conversion is an enzyme capable of cleaving the glucosidic bond between the isoflavone moiety of isoflavone glucoside and the glucose molecule. In a preferred embodiment, the enzyme is a saccharaidase or gluco-amylase enzyme capable of cleaving 1,4-glucosidic linkages. The enzyme may be present in soybean molasses, or may be added to the soy molasses material with a commercially available enzyme. In this specification, an enzyme originally present is referred to as a " residue enzyme " and an enzyme added to soybean molasses is referred to as a " supplement enzyme ".

Sufficient enzymes must be present to convert at least the majority, preferably almost all of the isoflavone glucosides to the aglucone isoflavones. In general, when the residual enzyme in the soy molasses material is insufficient to effect said conversion, a supplementary enzyme must be added to the soy molasses material. In a preferred embodiment, a supplementary enzyme is added to the soy molasses material irrespective of whether a sufficient residual enzyme is present in the soy molasses material because the addition of the supplementary enzyme results in an almost complete conversion of the glucoside to the aglucone Because the time required for the " When supplementary enzymes are added, the supplemental enzymes should be added so that the total enzyme concentration present in the soy molasses material is from about 0.1 to about 10% by weight of the solids in the soy molasses material, based on dry weight.

Supplementary enzymes are selected based on optimal activity and cost effectiveness under selected pH and temperature conditions. Supplementary enzymes are enzymes capable of cleaving the bond between the isoflavone moiety and the glucose molecule of isoflavone glucoside, such as saccharidase and gluco-amylase enzyme capable of cleaving the 1,4-glucosidic bond. Preferred supplementary enzymes are the commercially available alpha-and beta-glucosidase enzymes, beta-galactosidase enzymes, gluco-amylase enzymes and pectinase enzymes. Examples of particularly preferred enzymes include, but are not limited to, 100 l of biopectinase (preferably at a pH of about 3 to about 6), 300 l of biopectinase (optimal pH of about 3 to about 6), biopectinase OK (Optimum pH is about 3 to about 6), biolactase 30,000 (optimum pH is about 3 to about 6), and neural lactase (optimal pH is about 6 to about 8), all of which are commercially available from Florida 34243 Sarasota Post Office Box 3917 Available from Quest International, 57th Street, 1833. A particularly preferred enzyme is lactase F (optimum pH is about 4 to about 6) and lactase 50,000 (optimum pH is about < RTI ID = 0.0 > About 4 to about 6). Other particularly preferred complementary enzymes include G-Zyme G990 (optimal pH is about 4 to about 6) available from Enzyme Development Laboratories, Pen Plaza 2, New York Sweet 2412, New York, 10121, Lactase concentrate (optimum pH is about 4 to about 6); Lactozyme 3000L (optimum pH is about 6 to about 8) and Alpha-Gal 600L (optimum pH is about 4 to about 8) commercially available from Novo Nordisk Bio Industries Incorporated of Danbury Turner Road 33, About 6.5): camelact L2000 available from Giust Brocca des Food Ingestion Incorporated of King of Prussia, Pennsylvania, USA (optimum pH is about 4 to about 6); And Neutral lactase (the optimum pH is about 6 to about 8) available from the Pfizer Science Group, 42nd Street, New York East 10017 New York East.

The pH range for converting isoflavone glucoside to aglucone isoflavone is from about 3 to about 9. Since the pH used is mainly dependent on the type of enzyme used, it should be selected accordingly. Although the pH of the soy molasses material is thought to be lower during the conversion process, the residual enzyme is active within a pH range of about 7 to about 9. As described above in some specific enzymes, the complementary enzyme exhibits activity within the specified optimum pH range by the manufacturer of the enzyme. Typically, the complementary enzyme exhibits activity at a pH in the neutral pH range of about 6 to about 8 or in an acid pH range of about 3 to about 6.

The pH of the soy molasses material may be adjusted to a value suitable to effect conversion of isoflavone glucoside to aglucone isoflavone. In most cases, the addition of a suitable acid such as at least one of acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid, or any other desirable reagent will increase the pH to a relatively high or low level required to convert the isoflavone complex to isoflavone glucoside Decrease from basic pH to low pH.

The temperature range for the soy molasses material for conversion from glucoside to aglucone is from about 5 캜 to about 75 캜. Since the temperature has a great influence on the activity of the enzyme, the conversion rate is also affected by the temperature. The complementary enzyme may exhibit activity at 70 ° C or higher, for example, Alpha-Gal 600L exhibits activity at 75 ° C, but it is preferred to perform the conversion at a lower temperature to avoid inactivation of the enzyme. In a preferred embodiment, the conversion is carried out at about 35 캜 to about 45 캜.

The time required to convert the glucoside to the aglucone depends on the enzyme-related factors, in particular the concentration, temperature and pH of the enzyme system. In most cases, almost complete conversion can be achieved within 24 hours, but it is desirable to add a supplementary enzyme to significantly increase the reaction rate. The selected supplementary enzyme, enzyme concentration, pH, and temperature perform almost complete conversion within about 2 hours, and it is most desirable to perform complete conversion within about 1 hour.

The conversion of isoflavone glucoside to aglucone isoflavone is highly efficient, thus converting at least most preferably, preferably all present, glucosides to aglucones. The term " most " as used herein means a conversion of at least about 50%, and " almost all " means a conversion of at least about 80%, preferably at least about 90%.

After conversion from isoflavone glucoside to aglucone isoflavone, a cake of agglomerated isoflavone rich material can be separated from the molasses material. The soy molasses material is treated at the pH and temperature to separate most of the aglycon isoflavones from the soy molasses material during the separation process. In a preferred embodiment, the soy molasses material has a pH in the range of about 3 to about 6.5, most preferably about 4.5, and a temperature in the range of about 0 째 C to about 35 째 C, preferably 0 째 C to about 10 째 C, RTI ID = 0.0 > 4 C < / RTI > The pH of the soy molasses material can be adjusted using a suitable acidic or basic reagent as appropriate.

The separation may be carried out by conventional methods for separating solids from liquids. The agglomerated isoflavone rich cake is preferably separated by centrifugation or filtration as described above in connection with separating the isoflavone-rich cake from the soy molasses material.

Also, aglucone isoflavone rich materials can be prepared from isoflavone rich materials recovered from soybean molasses, and methods for recovering isoflavone rich materials from soybean molasses have been described. Water is added to the recovered cake of isoflavone-rich material to form a slurry of isoflavone-rich material. The slurry may use a higher solids content, but may be diluted such that the solids content is from about 6% to about 13%. The isoflavone complex in the slurry is then converted to isoflavone glucoside by treating the slurry under the same conditions as previously described when converting the isoflavone complex into isoflavone glucoside in soy molasses. Specifically, the slurry is treated at a pH of from about 6 to about 13.5, preferably at a pH of from about 9 to about 11, at a temperature of from about 2 DEG C to about 120 DEG C for about 15 minutes to several hours. The slurry may be treated at a pH of about 11 and a temperature of from about 5 째 C to about 50 째 C, preferably about 35 째 C, for about 30 minutes to about 1 hour, or a pH of about 9 and about 45 to about 75 째 C, Lt; RTI ID = 0.0 > 73 C < / RTI > for about 4 hours to about 6 hours. If desired, the isoflavone glycoside rich material can be separated from the slurry in a manner similar to the separation of the isoflavone rich material from soybean molasses as previously described.

The isoflavone glucoside in the slurry is then converted to aglucone isoflavone under the same conditions as previously described with respect to the conversion of isoflavone glucoside to aglucone isoflavone in the soy molasses material. Specifically, an enzyme capable of cleaving the glycosidic bond between the isoflavone portion of the isoflavone glucoside and the glucose molecule under suitable pH and temperature conditions for a time sufficient to convert the isoflavone glucoside to the aglucone isoflavone, The isoflavone glucoside in the slurry is contacted. Preferred enzymes, pH conditions, temperatures and times are as previously described. Agglomerated isoflavone rich material can be separated from the slurry in a manner similar to the separation of the isoflavone rich material from the previously described soybean molasses.

In addition, the aglucone isoflavone rich material can be prepared from an isoflavone glucoside rich material recovered from the soy molasses material, and the method for recovering the isoflavone glucoside rich material from the soy molasses material is as described above. Water is added to the recovered cake of isoflavone glucoside-rich material to form a slurry of isoflavone glucoside-rich material. Although a higher solids content may be used, it is desirable to dilute the slurry to make the solids content from about 6% to about 13%. The isoflavone glucoside in the slurry is converted to aglucone isoflavone in the same manner as described previously for the isoflavone glucoside rich slurry formed from the isoflavone rich slurry. The agglomerisoflavone rich material can be converted and then separated from the slurry in a manner similar to the separation of the isoflavone rich material from the previously described soybean molasses.

Experiment

The present invention is more specifically illustrated by the following examples. The following examples are illustrative and are not intended to limit the scope of the invention in any way.

As already pointed out, the soy material comprising soybean molasses contains the isosflavones genistein, daidzein and glycitein " family " of isoflavones bearing the corresponding glucosides, complexes and agglomerates, Include complex 6 " -OMalenistin, 6 " -OAc genistein, glucoside genistein and agluconenistein; The daidzein family contains a complex 6 " -OMalidide, 6 " -OAc didezin, glucoside didezin and an agluconidase; In the following examples, the relative concentrations of isoflavones were calculated from the total concentration of the isoflavone group or from the isoflavones < RTI ID = 0.0 > of isoflavones < The total concentration of the isoflavones of the genistein group was the sum of 6 " -OMalgenistin, 6 " -OAc genistin, genistin and genistein concentrations, and the percentage of each of the isoflavones in the genistein group was Other genistein groups were determined relative to isoflavones:% Genistein + 6% -OMalgeneutin + 6% -OAc Genistin +% Genistein = 100%.

Example 1

In the first experiment, recovery of isoflavone-rich material from soybean molasses was investigated in soybean molasses at various concentrations. The total concentration of each isoflavone group was determined in a selected concentration of soy molasses sample, a cake separated from the soy molasses sample according to the method of the present invention, and a liquid in which the cake was removed by a separation procedure.

Soybean molasses was analyzed for the total concentration of all forms of isoflavones present. The soy molasses sample was diluted with water to have a solids content of 28% (1: 2 dilution), 13.7% (1: 4 dilution) and 6.6% (1: 8 dilution). The pH of all samples was adjusted to 4.5. The treated sample was then centrifuged at a rate of 3000 rpm for 30 minutes to separate the liquid faeces and cake portion from the sample. A set of samples was centrifuged at a temperature of 0.6 캜. Samples having 28% and 13.7% soy molasses solids were centrifuged at 60 ° C for comparison with samples having the same concentration of soy molasses solids separated at 0.6 ° C. The liquid and cake portions of the resulting sample were analyzed for concentrations of all forms of isoflavones present.

Table 1 below shows the concentration of isoflavones in various cakes and liquid fractions obtained in the test. The total concentrations presented represent the total amount of all forms of specific isoflavones, including complexes, glucosides and agglucones, and the units are expressed in terms of the amount of isoflavone (mg) per gram of cake or liquid fraction solids.

[Table 1]

In all isolated samples, the concentration of isoflavones was significantly higher in the cake than in the soy molasses starting material and was much higher than the isoflavone concentration in the liquid whey fraction solids. The samples isolated at 0.6 ° C contained a higher concentration of isoflavones in the cake than the corresponding samples isolated at 60 ° C with a higher concentration of isoflavones in the faecal fraction solids.

Example 2

In another experiment, the recovery of isoflavone glucoside rich material from soy molasses was investigated. The isoflavone complex in the soy molasses was converted to isoflavone glucoside and the isoflavone glucoside rich cake was isolated from the soy molasses material. Conversion rates were determined by quantitative reduction of percentages and concentrations of malonate and acetate esters of the isoflavone group in pairs with a corresponding quantitative increase in the percentage of glucosides in the same isoflavone group.

The soybean molasses starting material was analyzed for the concentration of the individual isoflavone compounds. Two samples of the soy molasses material were prepared by mixing the soy molasses with 1: 4 (100 g molasses + 300 g water) using water; And a dilution ratio of 1: 8 (50 g of molasses + 350 g of water). The pH of the sample was adjusted to 11, and the temperature of the sample was maintained at 35 DEG C for 30 minutes. Then, the pH of the sample was adjusted to 4.5, and the temperature was adjusted to 4 캜. The samples were centrifuged at 10,000 rpm at 4 ° C to separate soy molasses samples into cakes and liquid fakes. The concentrations of the individual isoflavone compounds in the fakes and cakes were analyzed.

Table 2 below illustrates the ratios between the various forms of isoflavones produced by the conversion of the isoflavone complex to the isoflavone glucoside compared to the soybean molasses starter material. The isoflavone concentration was expressed in ppm in the sample and expressed as a percentage of the total amount of the particular isoflavones (total amount of complex, glucoside and agglomerate forms) in the liquid or cake portion.

[Table 2]

In all samples treated under the conditions for conversion from isoflavone complex to isoflavone glucoside, the percentage of isoflavone glucoside in both the cake and the liquid portion was significantly greater in the corresponding unconverted soy molasses samples , The percentage of the corresponding isoflavone complexes in the sample was significantly less, indicating that large amounts of the isoflavone complexes were converted to their glucoside form. Also, upon separation, a large amount of glucoside isoflavone is separated in the cake to form an isoflavone glucoside rich material, which can be determined from the relative concentrations of the soybean molasses starting material and the fake and cake portions of each sample.

Example 3

In another experiment, the conversion of isoflavones in soybean molasses to aglucone isoflavones was investigated. The isoflavone complex in soybean molasses was converted to isoflavone glucoside, and the isoflavone glucoside was subsequently converted to aglucone isoflavone. The conversion of isoflavone glucoside to aglucone isoflavone was determined by the corresponding quantitative increase in the percentage of aglucone in the same isoflavone group by quantitative reduction of the concentration of glucoside in the paired isoflavones.

The soybean molasses starting material was diluted 1: 4 with water, and the concentration of individual isoflavone compounds was analyzed. The pH of the molasses was then adjusted to 11. The soy molasses was maintained at room temperature for 1 hour to form a glyconide-rich soy molasses material. The glucoside-rich soy molasses material was analyzed for the concentration of individual isoflavone compounds. After adjusting the pH of the glyconide-rich soy molasses material to 4.5, four samples were prepared using this material. Then, enzymes were injected into each sample, and G-Zyme 990, 100 L of Biopectinase, 50,000 L-lactase and 600 L of Alpha-Gal were added to the above samples in an amount of 10% by weight of molasses solids. Subsequently, the sample was treated at 50 DEG C for 6 hours to form a soy molasses material rich in aglucone isoflavone. Then, the content of isoflavone was analyzed for soybean molasses rich in aglucone isoflavone.

Table 3 below illustrates the distribution of various forms of isoflavones formed by the tests described above. Isoflavone concentrations were expressed in ppm in the sample and expressed as a percentage of the total amount of specific isoflavones (total amount of complex, glucoside and agglomerate forms).

[Table 3]

The agglutinin isoflavone content of the enzyme treated samples was much greater than that of soy molasses and glucoside rich soy molasses material, indicating that the enzyme treatment resulted in the conversion of a substantial amount of glucoside isoflavone to aglucone isoflavone. Selection of the appropriate enzyme, enzyme concentration, pH and temperature for the conversion allows almost all of the isoflavone glucoside to be converted to aglucone isoflavone, which is confirmed by the isoflavone distribution in the lactase 50,000 and alpha-gal 600L samples .

Example 4

In a final experiment, the isoflavone content in the isoflavone rich material, the isoflavone glucoside rich material and the aglucone isoflavone rich material was investigated and the isoflavone distribution in these materials was compared. Soybean molasses was diluted 1: 4 with water. Samples of diluted soy molasses were adjusted to pH 4.5, cooled to 0.6 ° C for 30 minutes in an ice bath, and centrifuged at 3000 rpm for 30 minutes to separate the cake of isoflavone rich material. The remaining diluted soy molasses was then adjusted to pH 11 with sodium hydroxide and treated at 50 DEG C for 1 hour to convert the isoflavone complex in the molasses to isoflavone glucoside. The pH of the molasses sample enriched in the glucosides was adjusted to 4.5 and cooled to 0.6 캜 for 30 minutes in an ice bath. Then, the mixture was centrifuged at a speed of 3000 rpm for 30 minutes to separate the cake made of the material rich in isoflavone glucoside. The remaining isoflavone glucoside rich soy molasses material was adjusted to pH 4.5 and the enzyme G-Zyme 990 was added to the material at a concentration of 2.6 g enzyme / 100 g molasses material. The enzyme and isoflavone glucoside rich soy molasses material was then treated at 50 DEG C for 18-20 hours to convert isoflavone glucoside to aglucone isoflavone. A sample of agaricin isoflavone-rich soy molasses material was cooled to 0.6 ° C for 30 minutes in an ice bath and centrifuged at a speed of 3000 rpm for 30 minutes to separate a cake made of agglomerated isoflavone-rich material. The recovered cake consisting of the isoflavone rich material, the isoflavone glucoside rich material and the aglucone isoflavone rich material was then examined for the isoflavone content.

Table 4 below shows the distribution of isoflavones in a cake consisting of an isoflavone rich material, an isoflavone glucoside rich material and an aglucone isoflavone rich material. The isoflavone distribution was expressed as a percentage of the total amount of specific isoflavones (total amount of complex, glucoside and aglycon forms).

[Table 4]

The efficiency of the conversion step can be confirmed by the isoflavone distribution of the material. The isoflavone glucoside rich material possessed an isoflavone rich material and an isoflavone content substantially consisting entirely of isoflavone glucoside, including a significantly greater amount of isoflavone glucoside than the aglucone isoflavone material. The agglomerated isoflavone-rich material contained significantly greater amounts of aglucone isoflavone than the isoflavone glucoside-rich material and the isoflavone-rich material, retaining an isoflavone content substantially consisting of aglucone isoflavone .

In the above example, the 6 " -OMal-genistin, 6 " -OAc-genistin, 6 " -OMal-didezin, 6 " -OAc- Isoflavones are prepared by dissolving 0.75 g of a sample (spray-dried or milled powder) and 80/20 methanol / water (water-insoluble powder) in a soybean product. The mixture was shaken using an orbital shaker for 2 hours at room temperature. After 2 hours, the remaining undissolved material was removed by filtration through Watson # 42 filter paper. 5 ml of the filtrate was diluted with 4 ml of water and 1 ml of methanol.

The extracted isoflavones were separated by HPLC (high performance liquid chromatography) using a Hewlett Packard C18 hyper silica reverse phase column. Isoflavone was injected into the column and elution was initiated by solvent gradient elution with 88% methanol, 10% water and 2% glacial acetic acid, and terminated with 98% methanol and 2% glacial acetic acid. 6 "-O-acetyl genistein, 6" -O-malonyl genistein, genistein, daidzin, 6 "-O-acetylidezine, 6" -O- Malonylidadezin, daidzein, glycitin and its derivatives and glycitein - were clearly analyzed. Peak detection was carried out by UV absorbance at 260 nm. Identification of the peaks was performed with an HPLC-mass spectrometer.

Quantitation was performed using pure standards (genistein, genistein, daidzein and daidzein) obtained from Indian Fine Chemicals Company, located in Somerville, NJ, USA. Reaction factors (integrated area / concentration) were calculated for each of the above compounds and used to quantify unknown samples. Since there is no pure standard available for the complex form, the reaction factors were estimated to be parent molecules, but the molecular weight differences were corrected. The response factors for glycitin were estimated to be of corrected genistein for molecular weight differences. The method provides a method for quantitation of individual isoflavones. Conveniently, it can be calculated as total genistein, total dideazine and total glycitein, which means the aggregate weight of these compounds, if all the complex forms are converted to their respective non-complex forms. These total amounts may also be determined directly by methods using acid hydrolysis to convert the complex form.

The foregoing is merely a preferred embodiment of the present invention. Modifications of the invention may be made without departing from the scope of the appended claims, which shall be construed in accordance with the principles of patent law, including the doctrine of equivalents.

In accordance with the present invention there is provided a method of reducing the severity of symptoms (e. G., Menopausal or premenstrual syndrome) caused by decreased cardiovascular risk factors, decreased levels of endogenous estrogens and alterations, For example, isoflavone-rich materials that can be used to inhibit growth of breast cancer cells and prostate cancer cells can be recovered from inexpensively available soy molasses.

Claims (4)

- providing a soy molasses material containing isoflavones; And - separating the isoflavone material from the soy molasses material at a temperature of 0 ° C to 35 ° C 1. A method for obtaining an isoflavone material from soybean molasses comprising: Wherein said isoflavone material comprises glycitin or glycitein. The method of claim 1, wherein said isoflavone material is separated from said molasses material at a pH of from 3.0 to 6.5. Claims 1. A health supplement composition comprising a solid material separated from a soy molasses material, wherein the solid material comprises at least two isoflavones, and at least one of the isoflavones of the solid material is glycitein, Wherein the solid substance is a health supplement. 4. The health supplement according to claim 3, wherein at least one of the isoflavones of the solid substance is isoflavone glucoside.